Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses

Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif ‘QALGGH’ were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.


Introduction
Pinus massoniana, a widely distributed coniferous tree in southern China, is not only an economically important species for timber, wood pulp, and rosin but also plays a significant ecological role in the forest ecosystem [1].The occurrence of seasonal soil drought in Southern China poses a significant natural constraint on the production and growth of Masson pine [2].In addition, the prevalence of pine wood disease in China over the past four decades has caused a tremendous disaster for coniferous plants.Therefore, it is highly necessary to cultivate drought-resistant and disease-resistant Masson pine.A large number of transcription factors (TFs) were involved in the response to biotic and abiotic stresses [3][4][5][6][7][8][9][10][11][12][13].Recent studies regarding comprehensive genome-wide sequencing, various transcriptome analyses, and the functional identification of numerous genes have demonstrated the involvement of various transcription-factor families in conferring abiotic stress tolerance in plants, including MYB [4,5], AP2/ERF [7], NAC [8], bHLH (basic helixloop-helix) [10], bZIP (basic leucine zipper) [11], homeodomain [13], WRKY [14], HSF [15], MADS-box [16], and zinc finger proteins [17].The large and diverse zinc finger protein family plays important roles in all aspects of plant growth and development [18].The first zinc finger protein (TF IIIA) was initially observed in Xenopus laevis oocytes in 1985, and the term 'zinc finger' was coined to describe their distinctive structural resemblance to fingers [19].Since then, zinc fingers with a variety of functions have been found in animals, plants, yeasts, and viruses [20].
Zinc finger proteins (ZFPs) play a critical role in transcriptional regulation, RNA binding, the regulation of apoptosis, and protein-protein interactions [21].They can be classified as C2H2, C2C2, C2HC, C8, C2HC5, C3HC4, CCCH, C6, C4, and C4HC3 [22,23].Among these types, C2H2-type zinc finger proteins are widely recognized as one of the most abundant and extensively investigated protein families in eukaryotes [8].C2H2 ZFPs contain two cysteine (C) and two histidine (H) residues in each finger, and these residues bind a zinc ion that stabilizes the ZFP and specifically binds to a domain within the promoter of the gene it regulates [24].In plants, numerous C2H2-ZFPs exhibit conserved structural features among themselves.Its sequence is characterized by C-X 2-4 -C-X 12 -H-X 3-5 -H (X represents any amino acid) [25].In multiple-fingered proteins, the adjacent fingers are separated by a long spacer that is highly variable in length and sequence from each other [21].Most fingers have a six-amino acid stretch, 'QALGGH', at a position corresponding to the N-terminal part of the recognition helix.They are called Q-type C2H2 ZFPs [26].
Currently, an increasing number of Q-type C2H2 ZFPs have been identified in various plants.Arabidopsis thaliana has been reported to possess a total of 176 C2H2 ZFPs [27], while 189, 109, 321, 54, and 47 C2H2 ZFPs have been identified in rice (Oryza sativa) [28], poplar (Populus trichocarpa) [29], soybean (Glycine max) [30], apple (Malus domestica Borkh.)[31] and wheat (Triticum aestivum) [32], respectively.Recent functional analysis has shown that C2H2-ZFPs are involved in regulating multiple growth-development processes and resisting biotic and abiotic stress in plants [12,20].EPF1 was identified from Petunia as the first plant-specific ZFP that interacted with the promoter region [33].Later, WZF1 was reported in wheat as a DNA-binding zinc finger protein that interacts with a cis element of histone genes [34].C2H2-ZFP245 might function as a downstream component of CBF/DREB proteins by repressing the expression of some genes in response to cold or drought stress in O. sativa [35].The overexpression of C2H2-SlZF3 in Solanum lycopersicum and A. thaliana resulted in the accumulation of AsA, thereby alleviating the oxidative damage caused by salt stress through the enhancement of the transgenic plants' ROS-scavenging capacity [36].In Glycine max, C2H2-GmSCOF-1 was induced by low temperature and abscisic acid (ABA) treatments but not by dehydration or high salinity [37].These results suggest that C2H2-ZFPs act as transcriptional activators or repressors in different stress signal-transduction pathways to regulate the transcriptional levels of downstream genes.
However, the identification of C2H2-ZFPs and stress-related functional analysis in P. massoniana is still lacking.In recent years, the advancements in transcriptome and genome analysis have provided us with an opportunity to identify TF families in plants that play important roles in diverse stress responses.In this study, 57 PmC2H2-ZFPs were identified from three stress-related transcriptomes and characterized using bioinformatic analysis.Then, the expression profiles of Q-type PmC2H2 in different tissues and stresses were studied to better understand their roles in regulating P. massoniana stress response.The results of this study provide insights into the characterization of PmC2H2-ZFPs, as well as screening a potential regulatory gene in response to biotic and abiotic stresses in P. massoniana.

Transcriptome-Wide Identification and Analysis of C2H2 ZFPs in P. massoniana
After excluding incomplete and highly homologous sequences (>97%), further confirmation of the conserved domain was obtained through prediction from SMART and CDsearch.We identified 57 C2H2 ZFPs with a highly conserved C2H2 domain (e-value < 0.001) from three transcriptomes in response to biotic and abiotic stresses in P. massoniana (Table S1).We designated them as PmC2H2-1 to PmC2H2-57 (Table 1).These C2H2 ZFPs exhibited significant variations in length, ranging from 239 to 725 amino acids (Aa), with an average length of 477.34 aa.The molecular weight (MW) of PmC2H2s ranged from 27.41 kDa to 81.74 kDa, while the isoelectric point (pI) values ranged from 4.82 to 9.46.Subsequently, subcellular localization prediction results revealed that the majority of PmC2H2s were localized in the nucleus, except for one instance where PmC2H2-29 was found to be localized in the chloroplast.

Phylogenetic Analysis and Domain Analysis of PmC2H2s
A phylogenetic tree was constructed using the ML method, as shown in the left part of Figure 1, which facilitated the classification of these 57 PmC2H2 ZFPs into five distinct subgroups denoted as 'I' to 'V'.Among them, group I represents the largest subgroup, comprising a total of 33 C2H2-ZFP members.The smallest group was Group III, which consisted of PmC2H2-8 and PmC2H2-43.Groups II, IV, and V contain 11, 8, and 3 members, respectively.According to the MEME program, for the identification of the conserved motifs of 57 C2H2 ZFPs (Figure 1), the amino acid length of the 10 motifs ranged from 8 to 50 (Table 2).We found that all members had at least one highly typical conserved C2H2 domain (motif 1).Motif 2 was distributed in all groups.In addition, motifs 5 and 7 were only found in group I. Motif 3 and motif 6 were found in six members in group III with an exception in group I (PmC2H2-12).In addition, we found that six members (PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33) contained the 'QALGGH' domain in group IV; they are also called Q-type C2H2 ZFPs.The occurrence of motif 4 was frequently observed in groups I and II.Motif 8 was distributed in all groups except Group V. Motif 9 occurs primarily in group I, with an exception in group II (PmC2H2-52).Motif 10 was distributed in group II and group V.In order to further determine the type and distribution of the C2H2 domains, a regular expression search showed that the most common C2H2 domain is C-X 2 -C-X 12 -H-X 3 -H, accounting for 49% of all C2H2, followed by C-X4-C-X12-H-X3-H and C-X4-C-X12-H-X4-H, accounting for 19% and 14%, respectively.However, the structure of C-X3-C-X12-H-X3-H and C-X3-C-X12-H-X5-H have not been found in Masson pine (Table S3).Considering that Q-type C2H2 is unique to plants and is widely involved in plant responses to both biotic and abiotic stresses, the functional studies of zinc finger proteins have predominantly focused on Q-type C2H2 members.However, there are no studies of Q-type C2H2 ZFPs that have been reported on Masson pine yet.Therefore, we selected these six Q-type members for further study (Table S2).Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.Table 2. Sequences of the 10 motifs of PmC2H2 ZFPs.

Expression Patterns of PmC2H2 ZFPs under Nematode and Drought Stress
Drought and nematodes are the two most important factors currently limiting the growth and development of the Masson pine.Therefore, two heatmaps were generated to analyze the expression levels of PmC2H2 ZFPs under nematode infestation and drought stress conditions, based on the transcriptome data (Figure 3).The transcriptome data represent gene-expression patterns in a specific temporal and spatial context, so only 34 and

Expression Patterns of PmC2H2 ZFPs under Nematode and Drought Stress
Drought and nematodes are the two most important factors currently limiting the growth and development of the Masson pine.Therefore, two heatmaps were generated to analyze the expression levels of PmC2H2 ZFPs under nematode infestation and drought stress conditions, based on the transcriptome data (Figure 3).The transcriptome data represent gene-expression patterns in a specific temporal and spatial context, so only 34 and 22 expression patterns of PmC2H2 ZFPs were obtained from the RNA-seq transcriptomics data, respectively.After infection by nematodes, 18 members of the C2H2 family showed induced expression at different time points.The expression of 5 members was suppressed after nematode infection, while the expression levels of 11 members initially increased and then decreased over time.As the drought intensifies, the expression levels of 15 members tend to increase, while the expression levels of 7 members decrease.

Expression Patterns of Q-Type C2H2 ZFPs Genes in Different Tissues
The qRT-PCR analysis in Figure 4 showed the expression patterns of six Q-type PmC2H2s in eight different tissues: shoot apices (T); young needle leaves (YL); old needle leaves (OL); young stem (YS); old stem (OS); xylem (X); phloem (P); and root (R).It is evident from the results that PmC2H2-4, PmC2H2-5, and PmC2H2-20 exhibit high expression in needles.PmC2H2-16 and PmC2H2-20 expressed significantly in the root, while PmC2H2-33 expressed highly in the young stem.The expression levels of PmC2H2-4 and PmC2H2-5 in the phloem are extremely low, while PmC2H2-20 and PmC2H2-24 have the lowest expression in the xylem.In addition, PmC2H2-33 exhibited a low expression in the old stem.Notably, the expression of PmC2H2-16 in stems and the xylem was almost undetectable.

Expression Patterns of Q-Type C2H2 ZFPs Genes in Different Tissues
The qRT-PCR analysis in Figure 4

Expression Levels of Q-Type C2H2 ZFPs Genes under Abiotic Stresses
Figure 5 shows the expression patterns of six genes under different abiotic stresses.PmC2H2-20 and PmC2H2-24 were induced significantly by ABA treatment (Figure 5a), while other genes exhibited lower expression levels under ABA treatment.Under natural drought conditions, with the exception of PmC2H2-20 which showed suppressed expression on the 20th day of drought, other genes were induced at different stages of drought.PmC2H2-20 and PmC2H2-33 were suppressed significantly by ETH (Figure 5c), and PmC2H2-5 was not sensitive to ETH treatment.The expressions of PmC2H2-4, PmC2H2-16, and PmC2H2-24 increased significantly at first and then decreased after 6 h.The expression of PmC2H2-5 and PmC2H2-16 were suppressed under H2O2 treatment, while other genes were induced by H2O2 first, and then, their expression presented a downward trend (Figure 5d).The PmC2H2-20 gene exhibited no sensitivity to mechanical injury stress (Figure 5e), while the expression levels of the remaining five genes displayed an initial increase followed by a subsequent decrease over time, with peak expression observed at either 3 h or 6 h.The expressions of PmC2H2-16, PmC2H2-20, and PmC2H2-33 were inhibited by MeJA (Figure 5f), and the expression levels of PmC2H2-24 increased significantly after MeJA treatment.The expression of PmC2H2-20 was significantly inhibited under salt stress (Figure 5g), and the expression changes of PmC2H2-5 under salt stress were not obvious.The expression levels of PmC2H2-20 and PmC2H2-33 significantly decreased after PEG treatment, while the other four genes were induced to varying degrees (Figure 5h).After SA treatment, it was worth noting that the expression level of PmC2H2-20 significantly increased within 3 h and then recovered.The expression levels of PmC2H2-20 were found to be significantly suppressed in most treatments, except for ABA and SA treatment, suggesting that PmC2H2-20 may play a significant role in stress resistance in Masson pine.

Expression Levels of Q-Type C2H2 ZFPs Genes under Abiotic Stresses
Figure 5 shows the expression patterns of six genes under different abiotic stresses.PmC2H2-20 and PmC2H2-24 were induced significantly by ABA treatment (Figure 5a), while other genes exhibited lower expression levels under ABA treatment.Under natural drought conditions, with the exception of PmC2H2-20 which showed suppressed expression on the 20th day of drought, other genes were induced at different stages of drought.PmC2H2-20 and PmC2H2-33 were suppressed significantly by ETH (Figure 5c), and PmC2H2-5 was not sensitive to ETH treatment.The expressions of PmC2H2-4, PmC2H2-16, and PmC2H2-24 increased significantly at first and then decreased after 6 h.The expression of PmC2H2-5 and PmC2H2-16 were suppressed under H 2 O 2 treatment, while other genes were induced by H 2 O 2 first, and then, their expression presented a downward trend (Figure 5d).The PmC2H2-20 gene exhibited no sensitivity to mechanical injury stress (Figure 5e), while the expression levels of the remaining five genes displayed an initial increase followed by a subsequent decrease over time, with peak expression observed at either 3 h or 6 h.The expressions of PmC2H2-16, PmC2H2-20, and PmC2H2-33 were inhibited by MeJA (Figure 5f), and the expression levels of PmC2H2-24 increased significantly after MeJA treatment.The expression of PmC2H2-20 was significantly inhibited under salt stress (Figure 5g), and the expression changes of PmC2H2-5 under salt stress were not obvious.The expression levels of PmC2H2-20 and PmC2H2-33 significantly decreased after PEG treatment, while the other four genes were induced to varying degrees (Figure 5h).After SA treatment, it was worth noting that the expression level of PmC2H2-20 significantly increased within 3 h and then recovered.The expression levels of PmC2H2-20 were found to be significantly suppressed in most treatments, except for ABA and SA treatment, suggesting that PmC2H2-20 may play a significant role in stress resistance in Masson pine.
stress were not obvious.The expression levels of PmC2H2-20 and PmC2H2-33 significantly decreased after PEG treatment, while the other four genes were induced to varying degrees (Figure 5h).After SA treatment, it was worth noting that the expression level of PmC2H2-20 significantly increased within 3 h and then recovered.The expression levels of PmC2H2-20 were found to be significantly suppressed in most treatments, except for ABA and SA treatment, suggesting that PmC2H2-20 may play a significant role in stress resistance in Masson pine.In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".

Transcriptional Activity Analysis
The transcriptional activity of Q-type C2H2 was analyzed.We successfully cloned PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 and constructed them into the PGBKT7 vector.The yeast strains that contained recombinant plasmids were cultured on the SD/-Trp medium first.Verified positive yeast colonies were collected onto the higher selective culture medium SD/-Trp/-His and SD/-Trp/-His/-Ala.The photographs of the yeast growth assay (Figure 6) showed that only PmC2H2-20 can grow on the severe selective medium (SD/-Trp/-His/-Ala) and activate the expression of reporter genes, resulting in the production of β-galactosidase, which turns substrate x-αgal blue, whereas the remaining five genes did not exhibit any self-activation.This result suggested that the PmC2H2-20 can act as a transcriptional activator by binding to the promoter sequence of downstream reporter genes, thereby activating their transcription and helping P. massoniana resist stress.

Discussion
The C2H2-type zinc finger proteins are widely distributed among eukaryotes and play significant roles in diverse biological processes, including hormone signaling, DNA In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".

Transcriptional Activity Analysis
The transcriptional activity of Q-type C2H2 was analyzed.We successfully cloned PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 and constructed them into the PGBKT7 vector.The yeast strains that contained recombinant plasmids were cultured on the SD/-Trp medium first.Verified positive yeast colonies were collected onto the higher selective culture medium SD/-Trp/-His and SD/-Trp/-His/-Ala.The photographs of the yeast growth assay (Figure 6) showed that only PmC2H2-20 can grow on the severe selective medium (SD/-Trp/-His/-Ala) and activate the expression of reporter genes, resulting in the production of β-galactosidase, which turns substrate x-α-gal blue, whereas the remaining five genes did not exhibit any self-activation.This result suggested that the PmC2H2-20 can act as a transcriptional activator by binding to the promoter sequence of downstream reporter genes, thereby activating their transcription and helping P. massoniana resist stress.In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".

Transcriptional Activity Analysis
The transcriptional activity of Q-type C2H2 was analyzed.We successfully cloned PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 and constructed them into the PGBKT7 vector.The yeast strains that contained recombinant plasmids were cultured on the SD/-Trp medium first.Verified positive yeast colonies were collected onto the higher selective culture medium SD/-Trp/-His and SD/-Trp/-His/-Ala.The photographs of the yeast growth assay (Figure 6) showed that only PmC2H2-20 can grow on the severe selective medium (SD/-Trp/-His/-Ala) and activate the expression of reporter genes, resulting in the production of β-galactosidase, which turns substrate x-αgal blue, whereas the remaining five genes did not exhibit any self-activation.This result suggested that the PmC2H2-20 can act as a transcriptional activator by binding to the promoter sequence of downstream reporter genes, thereby activating their transcription and helping P. massoniana resist stress.Figure 6.Transcriptional activation assay of six PmC2H2 genes.Empty pGBKT7 vector was used as a negative control and pGBKT7-PmC3H20 was used as a positive control.

Discussion
The C2H2-type zinc finger proteins are widely distributed among eukaryotes and play significant roles in diverse biological processes, including hormone signaling, DNA Figure 6.Transcriptional activation assay of six PmC2H2 genes.Empty pGBKT7 vector was used as a negative control and pGBKT7-PmC3H20 was used as a positive control.

Discussion
The C2H2-type zinc finger proteins are widely distributed among eukaryotes and play significant roles in diverse biological processes, including hormone signaling, DNA or RNA binding, and stress response [22,[38][39][40].Moreover, extensive exploration has been conducted to identify C2H2-type zinc finger proteins in various plant species.However, the lack of a reference genome for P. massoniana has created a knowledge gap in the comprehensive identification of C2H2 TF families in P. massoniana at the genomic level.Therefore, we can only temporarily fill this gap by using transcriptome data to identify stress-response TFs, thereby establishing a foundation for future research.
A total of 57 PmC2H2 ZFPs were identified from three transcriptomes and divided into five subgroups.Almost all PmC2H2 ZFPs were predicted to localize in the nucleus, supporting their role as a transcription factor in the nucleus.In addition, we confirmed the subcellular localization of PmC2H2-4 and PmC2H2-20 in tobacco leaves through instantaneous transformation, which is consistent with the prediction.In yeast and A. thaliana, C2H2 ZFPs were classified based on the quantity and configuration of ZF domains [41].The C2H2 sequence is especially characterized by C-X 2,4 -C-X 12 -H-X 4-5 -H in P. massoniana.However, it lacks the forms of C-X 3 -C-X 12 -H-X 3 -H and C-X 3 -C-X 12 -H-X 5 -H compared to other plants.The majority type of conserved C2H2 domains exhibited in P. massoniana are C-X 2 -C-X 12 -H-X 3 -H.
In addition, previous researchers have demonstrated that the surrounding residues at the C-terminus of the 'QALGGH' motif play a pivotal role in facilitating specific DNA recognition [42].The AGC(T) sequence was identified as the optimal binding site for the first zinc finger (ZF) of the Petunia ZPT2-2 protein, while the CAGT core was determined to be the preferred binding site for the second ZF [43].This discovery confirms that flanking residues play a pivotal role in determining specificity for recognizing DNA target sequences and facilitating effective DNA binding [44].We also identified six Q-type C2H2 ZFPs from Masson pine, all of which possess a 'QALGGH' motif.Current research has shown that Q-type C2H2 ZFPs are widely involved in plant biotic and abiotic stress responses.In addition, the expression of C2H2 ZFP is modulated by tissue-specific variations and abiotic stresses [45].Consequently, we further investigated the expression patterns of six Q-type C2H2s under diverse stresses and tissues.
The role of C2H2 ZFPs as transcription factors in response to abiotic stresses has been extensively studied across various plant species [46].The upregulation of a series of resistance genes can be induced by ABA, leading to their enhanced expression under conditions of drought and salt stress.This mechanism enhances the plant's capacity to endure osmotic stress [47,48].STZ/ZAT10 derived from A. thaliana can serve as a supplementary factor for yeast calcineurin mutants, enhancing salt tolerance in transgenic yeast [21,49,50].The expression of LkZFP6 in Larix kaempferi can be induced by ABA treatment [51].The qRT-PCR results revealed that PmC2H2-20 and PmC2H2-24 can be induced by ABA treatment, indicating that they may regulate Masson pine in response to abiotic stresses through the ABA-mediated signal pathway.
It is worth noting that PmC2H2-20 exhibits significant inhibition under nematode stress and treatments other than ABA and SA, indicating its involvement in the biotic and abiotic stress response, such as drought, H 2 O 2 , damage, MeJA, NaCl, and PEG.In addition, PmC2H2-24 can be induced by all treatments and nematode infection, suggesting its potential involvement in response to external stresses in P. massoniana.The stressinduced response pattern of the PmC2H2-4 closely resembles that of PmC2H2-24, with the exception that its expression level is repressed upon ABA treatment.The remaining three Q-type C2H2 ZFPs exhibited distinct expression patterns in response to multiple stresses, implying the involvement of Q-type C2H2 genes in response to abiotic stresses.
The tissue-specific expression of genes usually is preliminarily used to predict their corresponding functions [52].Previous research reported that the expression profiles of plant C2H2-ZFPs vary in different tissues and abiotic stresses [12,45].A tissue-specific expression analysis showed that PmC2H2-4, PmC2H2-5, and PmC2H2-20 may perform specific functions in needles.Whereas PmC2H2-16 and PmC2H2-24 function in the root, PmC2H2-33 functions in the young stem.A previous report showed that ZAT18 (AT3G53600) played a positive role in drought tolerance in Arabidopsis [53].PmC2H2-16 and PmC2H2-24, which are induced by drought, may be correlated with root growth and drought tolerance in P. massoniana.
In this study, we found the transcriptional activation of PmC2H2-20 in the yeast system, indicating that PmC2H2-20 could activate the expression of downstream reporter genes.The C2H2-type zinc protein of A. thaliana has been demonstrated in relevant studies to serve as a pivotal regulator of ROS signaling, thereby functioning as an integrative factor for coordinating responses to diverse abiotic stresses [54].
Although the role of C2H2 ZFPs has been extensively studied in model plants, such as Arabidopsis and Oryza sativa, there is little research on their involvement in pinaceae's tolerance to both biotic and abiotic stresses.Our study has successfully identified 57 C2H2 ZFPs from the transcriptomes of Masson pine for the first time and analyzed their expression patterns under two major stress conditions currently restricting the development of P. massoniana.Furthermore, we have thoroughly investigated the expression patterns of Q-type C2H2 members under various abiotic stresses and screened a potential candidate gene, PmC2H2-20, which may play a crucial role as an important regulatory factor in the stress response of P. massoniana.These findings provide insights for future functional studies on the C2H2 genes involved in stress resistance mechanisms in P. massoniana.

Subcellular Localization Analysis
The open reading frame (ORF) regions of PmC2H2-4 and PmC2H2-20 without a stop codon were linked with the pCAMBIA-1302-mGFP5 vector (primers were listed in Table S4).After PCR verification, the positive Agrobacterium was transferred to an LB medium supplemented with kanamycin (50 mg/L) and rifampicin (25 mg/L), followed by incubation until the optical density (OD) value reached 0.6.Subsequently, it was co-cultivated with the P19 (RNA Silencing Inhibitor) Agrobacterium strain in a suspension containing 150 µM acetosyringone, 10 mM MgCl2, and 10 mM 2-(N-morpholino) ethanesulfonic acid (MES).The leaves of 4-week-old Nicotiana benthamiana were injected with the mixed solution.Afterwards, the infiltrated N. benthamiana plants were kept in darkness for 48 h.The LSM710 confocal microscope (Zeiss, Jena, Germany) was used to capture the GFP signal.

Plant Materials and Abiotic Stress Treatments
Two-year-old, healthy, and uniformly sized seedlings of P. massoniana were selected for tissue-specific analysis and abiotic treatments.The drought treatment was performed as follows.Needles were collected five times over a period of 20 days, allowing for natural evaporation after watering at day 0. The samples were collected at five time points with the field capacity as follows: 0 d (67%); 3 d (63%); 7 d (58%); 12 d (46%); and 20 d (34%).The osmotic stress was induced by soaking the seedlings in the 15% polyethylene glycol (PEG6000) solution and 200 mM NaCl solution.The mechanical damage treatment method was performed by cutting the upper half of the needles.For plant hormones treatment, the selected seedlings were sprayed independently with 100 µM ABA (abscisic acid); 1 mM SA (salicylic acid); 10 mM H 2 O 2 (hydrogen peroxide); 10 mM MeJA (methyl jasmonate); and 50 µM ETH (ethephon) solutions (50 mL) on the surface of needles.Afterward, needles were sampled at 0 h, 3 h, 6 h, 12 h, and 24 h after treatment.All treatments were conducted on three biological replicates.

RNA Extraction and qRT-PCR Analysis
The total RNA of P. massoniana was extracted following the protocol provided in the FastPure plant total RNA isolation kit (RC401, Vazyme Biotech, Nanjing, China).The RNA concentration and purity were measured with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA integrity was estimated by 1% agarose gel electrophoresis.First-strand cDNA was synthesized using the One-step gDNA Removal and cDNA Synthesis Kit (AT311, TransGen Biotech, Beijing, China).Primers for quantitative real-time reverse transcription PCR (qRT-PCR) were designed using Primer 5.0 (Table S4).SYBR Green reagents were used to detect the target sequence.Each PCR mixture (10 µL) contained 1 µL of diluted cDNA (20× dilution), 5 µL of SYBR Green Master Mix (11184ES03, Yeasen Biotech, Shanghai, China), 0.4 µL of each primer (10 µM), and 3.

Transcriptional-Activation Activity Assay
The open reading frames of PmC2H2-4, PmC2H2-5, PmC2H2-16, PmC2H2-20, PmC2H2-24, and PmC2H2-33 were fused with the pGBKT7 vector (primer sequences were listed in Table S4).Subsequently, these recombinant plasmids were transformed into AH109 yeast strains (YC1010, Weidi Biotech, Shanghai, China), which were cultured on the SD/-Trp medium at 29 • C for 48 h.Verified positive yeast colonies were collected into 200ul ddH 2 O. Subsequently, a repetitive procedure was performed to withdraw 5 µL from the suspension and deposit it onto the culture medium SD/-Trp/-His/-Ade, which contained X-α-Gal as an indicator.The empty pGBKT7 was used as a negative control, and PmC3H20, previously confirmed to exhibit transcriptional self-activation, was employed as a positive control [17].

Conclusions
In this study, we identified a total of 57 C2H2 ZFPs in P. massoniana and performed a bioinformatics analysis.These members were classified into five subgroups and six Q-type C2H2 ZFPs were selected for further investigation to explore their expression patterns under various stress conditions and in different tissues.Subcellular localization prediction results revealed nuclear localization for all genes except PmC2H2-29, while experimental validation confirmed the nuclear localization of PmC2H2-4 and PmC2H2-20.Two heatmaps showed that PmC2H2s could respond to pine wood nematodes stress and drought, respectively.However, we noticed that the expression levels of PmC2H2-20 are down-regulated, except for its induction under ABA stress.Furthermore, the transcriptional activation activity of PmC2H2-20 was detected, indicating its role as a potential transcriptional activator involved in stress-response mechanisms.This study establishes a theoretical basis for further investigation into C2H2 TFs associated with pine wood nematode and drought resistance in P. massoniana, while also presenting PmC2H2-20 as a potential candidate gene that exhibits significant responsiveness to stress.

17 Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with different colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Figure 1 .
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

3 .
Subcellular Localization AnalysisSubcellular localization prediction indicated that most C2H2 TFs likely exert regulatory functions at the nucleus.In order to further comprehend the localization features of PmC2H2 ZFPs, PmC2H2-4 and PmC2H2-20 were selected for the experiment.A fluorescent signal was found after transient transformation in tobacco leaves.The GFP signal was distributed throughout the whole cell in the control; however, mGFP5 fused with PmC2H2-4 and PmC2H2-20 only showed fluorescence in the nucleus (Figure2).The results indicated that PmC2H2-4 and PmC2H2-20 are nuclear localization proteins.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 172.3.Subcellular Localization AnalysisSubcellular localization prediction indicated that most C2H2 TFs likely exert regulatory functions at the nucleus.In order to further comprehend the localization features of PmC2H2 ZFPs, PmC2H2-4 and PmC2H2-20 were selected for the experiment.A fluorescent signal was found after transient transformation in tobacco leaves.The GFP signal was distributed throughout the whole cell in the control; however, mGFP5 fused with PmC2H2-4 and PmC2H2-20 only showed fluorescence in the nucleus (Figure2).The results indicated that PmC2H2-4 and PmC2H2-20 are nuclear localization proteins.

Figure 2 .
Figure 2. Subcellular localization analysis of PmC2H2-4 and PmC2H2-20 proteins in N. benthamiana leaves.The scale in the images is 20 µm.pCAMBIA-1302-mGFP5 was the control.DAPI-4′,6-diamidino-2-phenylindole, a blue fluorescent dye that shows DNA location.Chloroplast-chloroplast auto-fluorescence, displays the location of chloroplasts.GFP-green fluorescence protein, displays the location of the target protein.Bright-bright field.Merged-merged picture of four overlapped channels.

Figure 2 .
Figure 2. Subcellular localization analysis of PmC2H2-4 and PmC2H2-20 proteins in N. benthamiana leaves.The scale in the images is 20 µm.pCAMBIA-1302-mGFP5 was the control.DAPI-4 ′ ,6diamidino-2-phenylindole, a blue fluorescent dye that shows DNA location.Chloroplast-chloroplast auto-fluorescence, displays the location of chloroplasts.GFP-green fluorescence protein, displays the location of the target protein.Bright-bright field.Merged-merged picture of four overlapped channels.
showed the expression patterns of six Q-type PmC2H2s in eight different tissues: shoot apices (T); young needle leaves (YL); old needle leaves (OL); young stem (YS); old stem (OS); xylem (X); phloem (P); and root (R).It is evident from the results that PmC2H2-4, PmC2H2-5, and PmC2H2-20 exhibit high expression in needles.PmC2H2-16 and PmC2H2-20 expressed significantly in the root, while PmC2H2-33 expressed highly in the young stem.The expression levels of PmC2H2-4 and PmC2H2-5 in the phloem are extremely low, while PmC2H2-20 and PmC2H2-24 have the lowest expression in the xylem.In addition, PmC2H2-33 exhibited a low expression in the old stem.Notably, the expression of PmC2H2-16 in stems and the xylem was almost undetectable.sion in needles.PmC2H2-16 and PmC2H2-20 expressed significantly in the root, while PmC2H2-33 expressed highly in the young stem.The expression levels of PmC2H2-4 and PmC2H2-5 in the phloem are extremely low, while PmC2H2-20 and PmC2H2-24 have the lowest expression in the xylem.In addition, PmC2H2-33 exhibited a low expression in the old stem.Notably, the expression of PmC2H2-16 in stems and the xylem was almost undetectable.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 17

Figure 4 .
Figure 4.The relative expression levels of six Q-type PmC2H2s in different tissues: shoot apices (T), young needle leaf (YL), old needle leaf (OL), young stem (YS), old stem (OS), xylem (X), phloem (P), and root (R).The relative expression level in 'T' was set as "1".The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b', and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters.

Figure 4 .
Figure 4.The relative expression levels of six Q-type PmC2H2s in different tissues: shoot apices (T), young needle leaf (YL), old needle leaf (OL), young stem (YS), old stem (OS), xylem (X), phloem (P), and root (R).The relative expression level in 'T' was set as "1".The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b', and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters.

Figure 5 .
Figure 5.The figure above shows the expression levels of five PmC2H2 genes under different abiotic treatments, namely (a) ABA, (b) drought, (c) ETH, (d) H2O2, (e) mechanical damage, (f) MeJA, (g) NaCl, (h) PEG, and (i) SA.The absence of any significant difference is indicated by the presence of identical lowercase letters across different columns.Conversely, the presence of completely distinct lowercase letters between different columns signifies a statistically significant difference (p < 0.05).In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".

Figure 6 .
Figure 6.Transcriptional activation assay of six PmC2H2 genes.Empty pGBKT7 vector was used as a negative control and pGBKT7-PmC3H20 was used as a positive control.

Figure 5 .
Figure 5.The figure above shows the expression levels of five PmC2H2 genes under different abiotic treatments, namely (a) ABA, (b) drought, (c) ETH, (d) H 2 O 2 , (e) mechanical damage, (f) MeJA, (g) NaCl, (h) PEG, and (i) SA.The absence of any significant difference is indicated by the presence of identical lowercase letters across different columns.Conversely, the presence of completely distinct lowercase letters between different columns signifies a statistically significant difference (p < 0.05).In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".

Figure 5 .
Figure 5.The figure above shows the expression levels of five PmC2H2 genes under different abiotic treatments, namely (a) ABA, (b) drought, (c) ETH, (d) H2O2, (e) mechanical damage, (f) MeJA, (g) NaCl, (h) PEG, and (i) SA.The absence of any significant difference is indicated by the presence of identical lowercase letters across different columns.Conversely, the presence of completely distinct lowercase letters between different columns signifies a statistically significant difference (p < 0.05).In cases where multiple lowercase letters are present within the same column, it implies that there is no significant difference between that particular column and other columns containing any one of those lowercase letters.The relative expression at 0 h is normalized to "1".
2 µL of ddH 2 O.The PCR program stages were (1) 95 • C for 2 min (preincubation); (2) 95 • C for 10 s, and (3) 60 • C for 30 s, repeated 40 times.The remaining steps use the instrument's default settings.The PCR quality was estimated based on the melting curves.The alpha-tubulin (TUA) gene was used as a reference gene [59].Three independent biological replicates and three technical replicates for each biological replicate were examined.Quantification was achieved using comparative cycle threshold (Ct) values, and gene-expression levels were calculated as 2(−∆∆Ct) [∆CT = CT Target − CT TUA.∆∆Ct = ∆Ct Target − ∆Ct CK].Duncan's test was used to examine the significance between different columns in IBM SPSS Statistics (Version 25).The lowercase letters represent the significance between different columns (p < 0.05).

Table 1 .
The characteristics of 57 C2H2 proteins identified and their subcellular localization prediction.
Figure 1.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with different colors represented different subgroups.The subgroup was named from "I" to "V".

Motif Length Motif Consensus Motif Logo 1 21 CEICGKGFSRPQNLQQHMRTH Figure 1. Phylogenetic
analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Table 2 .
Sequences of the 10 motifs of PmC2H2 ZFPs.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".

Table 2 .
Sequences of the 10 motifs of PmC2H2 ZFPs.Phylogenetic analysis and motif distribution of PmC2H2 ZFPs.Branches with differe colors represented different subgroups.The subgroup was named from "I" to "V".